Rapid identification and resistance assessment:

The future is mass spectrometry Dr Sanmarié Schlebusch

Director of Microbiology Mater Pathology

Brisbane

Outline Introduction

Plug and play

Pre-prep and post analysis

Other wonderful approaches

Other organisms

Closing statement

Introduction MS is broad spectrum – not restricted to pre-specified targets

Rapid

Masses of intact molecular ions and fragments easily determined by various types of MS

Tandem MS can produce fragment ions which provide information about molecular structures and sequences

Proteomics determines gene expression – does not focus on only a single gene

Protein expression may vary – but gene detection may not represent protein expression at all

MALDI-TOF MS

MS and Tandem MS (MS/MS)

MS

MS/MS

Plug and play MRSA – variable reports

VRE – routine use for VanB, fully automated

Beta-lactamases B. fragilis cfiA positive (MBL) vs cfiA negative Ampicillin resistant E. coli – poor reproducibility Clonal differentiation of resistant and susceptible clones cannot be expected for the majority of bacteria

Preparation and analysis required: Detection of enzymatic activity

Beta-lactamases Variety – difficult to propose universal primers MS detection of antimicrobial and degradation product Antimicrobial and organism+antimicrobial

Spectrum represents beta-lactam molecule, its salts or degradation products is analyzed Detect loss of molecule peaks and presence of hydrolysis products after couple of hours Described for ampicillin, piperacillin, cefotaxime, ceftazidime, ertapenem, imipenem and meropenem

Ampicillin after (A) incubation with the β-lactamase-negative E. coli strain DH5α (B) a β-lactamase-producing strain

(C) Inhibition of hydrolysis by a β-lactamase-producing strains was performed in the presence of clavulanic acid.

Nonhydrolyzed form of ampicillin peaks are highlighted in gray. Hydrolyzed form of ampicillin peaks are indicated with a small black arrow.

Hydrolysis of beta-lactam ring corresponds to a mass shift of +18Da

Detection of enzymatic activity

Carbapenem resistance

Phenotypic and genotypic testing issues with TAT, cost, specificity

MS – rapid, easy, readily available reagents Enterobacteriaceae, P. aeruginosa and B. fragilis – incubated with known MBLs and carbapenemases 67/70 carbapenemase producers correctly detected

Imipenem and degradation products Large proportion OXA type carbapenemases in A.baumannii

Ertapenem degradation

Ertapenem

+ NDM-1-carrying K. pneumoniae

+ IMP-1-carrying P. aeruginosa

Detection of enzymatic activity

Carbapenem resistance Hydrolysis of ertapenem by K. pneumoniae directly from positive blood cultures

Standardization of Acinetobacter carbapenemase detection (May 2013)

Comparative analysis of MBL and OXA type producing Acinetobacter strains resulted in reduction of imipenem peaks

Different effects was observed in different OXA type producers

MALDI-TOF MS spectra showing the most representative imipenem peaks

(A) Incubation solution only (B) Imipenem-cilastatin solution (C) OXA-24 like producing Acinetobacter added (D) IMP-8 Acinetobacter added

Representative comparison of the average intensities of the most representative peaks of imipenem (m/z 300 and 489) obtained after a 1-h incubation with the different

carbapenemases included in the study

Detection of enzymatic activity - conclusions

All studies similar results – this methodology has great potential to become routine

Principle of degradation product monitoring can be applied to other enzymatic resistance mechanisms

However, manual measurement and raw spectra analysis

For ertapenem and K.pneumoniae in positive blood cultures – MS calibrated to detect mass accuracy less than 0.5 Da

Interpretation and evaluation – expert knowledge required After peak assignment of sensitivity and resistance patterns – classification becomes easy

Special software and automation of acquisition and interpretation of results should be investigated and made available

Not able to detect other carbapenem resistance mechanisms. These would include porin alterations and efflux mechanisms for K. pneumoniae, P. aeruginosa and porin alterations, efflux mechanisms and PBP alterations for A. baumannii.

Preparation and analysis required Other

rRNA methyltransferase detection Purified ribosomes and purified enzymes Needs to be simplified to become routine

Porin detection OmpK36 porin in K. pneumoniae (responsible for penetration of carbapenems into periplasm) Failure to express OmpK36 due to an insertional inactivation or a nonsense mutation in the ompK36 gene Specific extraction followed by MALDI-TOF MS Porins detected on molecular weight

Carbapenem-resistant K.oxytoca ZC101; and K. pneumoniae Z5 and Z4. The arrows with dotted lines indicate the loss of 19,000- and 38,000-m/z peaks

representing OmpK36.

Other approaches Proteomic approaches

Entire protein complement expressed is studied Selective lysis and electrophoresis MALDI-TOF MS after SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis)

Described for outer membrane and periplasmic proteins altered in resistant isolates not routine

Other approaches Tandem MS

Kanamycin phosphorylation detection

Minisequencing DNA amplification, then allele-specific reactions measured by MALDI-TOF MS Specialized

Other approaches PCR-ESI MS

Measures m/z of PCR amplicons Described for antimicrobial resistance detection Detection of mixed bacteria and antimicrobial resistance detection eg. mecA or vanA/B (specific primers used) in IE

Nanoparticles MALDI-TOF MS not suitable for detection of small molecules due to matrix interference Nanoparticles for example magnetic nanoparticles can enrich and enhance detection of small molecules Improves signal to noise ratio

Other organisms Antifungal susceptibility

C. albicans and fluconazole Proteome monitored of Candida cells grown with and without antifungal

C. albicans and caspofungin CCI on standard Bruker Microflex used

Antiviral susceptibility RFMP assay based on MS analysis of small DNA fragments that include sites of mutation Distinct peaks relevant to each codon NRTIs, NNRTIs and PIs for HIV drug-resistance Fast, cost effective Bruker Biflex

Summary MALDI-TOF MS

Great potential Direct detection of antimicrobial resistance (plug and play) already in use Enzyme degradation of antimicrobials already in routine use Develop more methods Better software and automation

Other approaches Being investigated

Other resistance mechanisms Other MS and technology Different organisms

Final statement Proteome-level studies detect behavior of strains

Expression of proteins of interest Posttranslational modifications

Most routinely used molecular genetics are restricted to detection of known resistance determinants

Including WGS databases

Some proteomic analysis labor-intensive – reference centers

MALDI-TOF MS opens new avenues for clinical and experimental microbiology

© Daniel Zeevi | Apr 14, 2013

Then

NOW

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